Differential staining lowers the false positive detection in a novel volumetric measurement technique of microplastics

A novel method for the volumetric detection of microplastics in various environmental (soil, water) and food (fish, meat) matrices was developed. The method is based on the Nile Red staining of microplastics while eliminating probable interference by other organic polymers such as lignin, chitin, cellulosic materials, and other organic substances using a mixture of three water-based dyes (Calcofluor White, Evans Blue, and 4,6-diamidino-2-phenylindole [DAPI]). The excitation/emission ‘sweet spot’ was determined for water based blue dyes to detect them in a single channel for effective elimination of probable contaminations. Detection of microplastic particles using the Nile Red method was validated by comparing with traditional detection of microplastics via Fourier transform infrared spectroscopy (FTIR). Volumetric measurements of the microplastics present in environmental samples were made possible using Z-stack confocal microscopy images backed by threshold-based 3D segmentation. Regularly shaped microplastic materials were used to validate the volumetric measurement method. The proposed volumetric determination method will be very useful for screening microplastics in diverse media and improving the prevailing method using FTIR. • Nile Red staining for detecting environmental and food microplastics. • Removed most probable false detections using Calcofluor White, Evans Blue, and DAPI. • Volumetric measurement of microplastics from Z-stack CLSM images. • Regularly shaped microplastic materials were used to validate volumetric measurement.